Mice deficient in LMAN1 exhibit FV and FVIII deficiencies and liver accumulation of α1-antitrypsin

Abstract

The type 1-transmembrane protein LMAN1 (ERGIC-53) forms a complex with the soluble protein MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Mutations in either LMAN1 or MCFD2 cause the combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D), suggesting an ER-to-Golgi cargo receptor function for the LMAN1-MCFD2 complex. Here we report the analysis of LMAN1-deficient mice. Levels of plasma FV and FVIII, and platelet FV, are all reduced to ∼ 50% of wild-type in Lman1(-/-) mice, compared with the 5%-30% levels typically observed in human F5F8D patients. Despite previous reports identifying cathepsin C, cathepsin Z, and α1-antitrypsin as additional potential cargoes for LMAN1, no differences were observed between wild-type and Lman1(-/-) mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma. LMAN1 deficiency had no apparent effect on COPII-coated vesicle formation in an in vitro assay. However, the ER in Lman1(-/-) hepatocytes is slightly distended, with significant accumulation of α1-antitrypsin and GRP78. An unexpected, partially penetrant, perinatal lethality was observed for Lman1(-/-) mice, dependent on the specific inbred strain genetic background, suggesting a potential role for other, as yet unidentified LMAN1-dependent cargo proteins.

Figure 1

Characterization of mice carrying the Lman1 targeted allele. (A) Schematics of the cDNA structures of the WT murine Lman1 allele and the targeted allele with the gene-trap vector insertion. Locations of RT-PCR primers are indicated. (B) RT-PCR of total liver RNA isolated from Lman1^+/− and Lman1^−/− mice using 3 pairs of primers: F1-R1 indicates upstream of exons 10 and 11 junction; F2-R2, cross the exons 10 and 11 junction; and F3-R3, downstream of exons 10 and 11 junction. (C) Western blot analysis of liver lysates from WT (+/+), heterozygous (+/−), and LMAN1-deficient (−/−) mice using antibodies against LMAN1 and MCFD2. *Fusion protein of LMAN1 and β-GEO. MW indicates molecular weight.

Figure 2

FV and FVIII levels in Lman1 WT (+/+), heterozygote (+/−), and homozygous (−/−) mice. (A) Comparison of FVIII activities between WT and Lman1^−/− mice. FVIII activity was determined at ∼ 6 months of age by a 2-stage chromogenic assay. (B) Variation in FV activity for individual mice over a 6-week period. Blood was collected from each individual mouse at 2-week intervals, beginning at ∼ 6 months of age. FV activity was determined by a one-stage clotting assay. The group averages of each time points were plotted, with the 6-week average of FV levels in WT mice designated as 100%. (C) Relative FV antigen (%) in plasma (equal volume) and in platelets (equal number) as determined by Western blot analysis. The β-actin level in platelets serves as a loading control. The relative levels in different individual mice at ∼ 6 months of age were quantified and normalized against β-actin. Vertical bars in all panels represent SD.

Figure 3

Budding of COPII-coated vesicles is not affected by LMAN1 deficiency. Semi-intact WT and Lman1^−/− MEFs were permeabilized with digitonin and served as sources of the ER membranes. COPII proteins were supplied in rat liver cytosol. COPII-coated vesicles bud from the ER membrane with the addition of guanosine triphosphate and the adenosine triphosphate regeneration mix. The vesicles were purified by differential centrifugation and analyzed by Western blotting using the indicated antibodies.

Figure 4

ER morphology and expression of UPR markers in WT (+/+) and Lman1^−/− cells. (A) Color panels: Immunofluorescence staining of MEFs derived from WT and Lman1^−/− embryos. MEFs were fixed in 4% paraformaldehyde, incubated with polyclonal antibodies against ER TRAPα for the ER (red), KDEL receptor for the ERGIC (green), and giantin for the cis-Golgi (green in the first panel, red in the second panel). Black-and-white panels: Morphology of rough ER was visualized by electron microscopy in WT and Lman1^−/− hepatocytes. Scale bars represent 0.5μM. (B) Western blot analysis of GRP78 in liver lysates from WT and Lman1^−/− mice using anti-KDEL antibody. Each lane contains a sample from a different individual mouse.

Figure 5

Plasma and liver AAT levels. (A) AAT antigen is increased in Lman1^−/− mouse liver lysates. Liver lysates were prepared from WT and Lman1^−/− mice and analyzed for AAT, TRAPα, and β-actin by immunoblotting. Relative levels of the lower band of AAT were quantified and normalized to β-actin. (B) AAT accumulates in the ER of Lman1^−/− mouse liver. Liver lysates from WT and Lman1^−/− mice were digested with endo H and analyzed for AAT, albumin, CatZ, and β-actin by immunoblotting. Each lane contains a sample from a different individual mouse.